Nitric oxide synthase expression in the medullary respiratory related nuclei and its involvement in CO-mediated central respiratory effects in neonatal rats

The present study was conducted in order to observe the potential participation of the nitric oxide synthase-NO pathway in CO-mediated regulation of respiration of neonatal rats. An immunofluorescent histochemical technique was used to examine the existence of the neuronal nitric oxide synthase, a key enzyme of synthesizing NO, in medullary respiratory nuclei. The rhythmic respiratory-like discharges of hypoglossal rootlets of medullary slices were recorded to test the role of the nitric oxide synthase in CO-mediated respiratory effects. We observed neuronal nitric oxide synthase expressed in the medullary respiratory nuclei in conjunction with CO lengthened expiratory duration, decreased respiratory frequency, and increased inspiratory amplitude. These CO-mediated respiratory effects could be partially eliminated by prior treatment of the slices with Nω-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase. The results suggest that nitric oxide synthase-NO pathway might be involved in the CO-mediated central regulation of respiration at the level of medulla oblongata in neonatal rats.     

铅对培养海马神经细胞生长和nNOS表达的影响

目的研究不同剂量醋酸铅对原代培养的海马神经细胞生长和神经元型一氧化氮合酶(nNOS)表达的影响。方法将孕18dSD大鼠麻醉后剖腹取出胎鼠,采用海马神经细胞培养建立细胞模型,醋酸铅的染毒浓度分别为10-5mol/L(高剂量组)、10-7mol/L(中剂量组)、10-9mol/L(低剂量组),对照组给予等量培养液。染毒24h后用免疫组织化学法检测神经细胞球和单个神经细胞nNOS蛋白的表达。结果各剂量组对海马神经细胞胞体、轴突突起长度和细胞间连接均未见明显影响。高剂量组和中剂量组海马神经细胞球和散在的神经细胞nNOS的表达明显下降,与对照组相比差异具有统计学意义(P<0.05)。低剂量组海马神经细胞球和散在的神经细胞nNOS的表达较对照组有所下降,与对照组相比差异没有统计学意义(P>0.05)。结论低剂量铅下调培养的海马神经细胞nNOS的表达,这种下调作用具有剂量依赖性。     

Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.     

糖尿病性勃起功能障碍大鼠模型的建立及阴茎海绵体组织nNOS的变化

目的研究糖尿病性勃起功能障碍(ED)大鼠模型的建立及阴茎海绵体组织nNOS表达活性的变化。方法1.24只SPF级SD雄性大鼠,随机分为4组,按不同处理因素给大鼠腹腔注射链脲佐菌素(STZ),分别记录4d、1周、2周、3周的体质量、阿朴吗啡(APO)诱导阴茎的勃起次数及空腹血糖值。2.40只SPF级SD雄性大鼠,先随机分为两组,一组注射STZ后观察7周,另一组注射STZ后观察4周,按照成模标准,将每一处理组又分为对照组、成糖尿病性勃起功能障碍模型组、成糖尿病性非勃起功能障碍组、未成模组。采用免疫组化SP法检测大鼠阴茎组织中nNOS的表达,利用彩色图文分析系统,测量随机每高倍镜视野下累积光密度(IOD),以IOD值反映组织切片中nNOS表达程度。结果1.体质量及空腹血糖在四个不同时间点和不同处理组间均有显著性差异(P均<0.001),注射STZ60mg/kg组体质量较轻、血糖较高。APO诱导阴茎勃起次数在四个不同组间有显著性差异(F=2.831,P=0.046),注射STZ60mg/kg组应用APO后阴茎未勃起的例数较多。2.注射STZ后4周与注射STZ后7周处理组各不同组别间IOD存在显著性差异(F=3.864,P=0.020),糖尿病性勃起功能障碍组IOD均小于对照组、糖尿病非勃起功能障碍组及未成模组(P<0.05)。结论1.大鼠注射STZ后两周,血糖>7.2mmol/L及APO诱导阴茎未勃起的,可认为是糖尿病性勃起功能障碍模型(DM&ED);2.糖尿病ED大鼠阴茎海绵体组织中nNOS表达量明显减少,这可能是糖尿病性勃起功能障碍的发病机理之一。     

针刺对心理应激性勃起功能障碍大鼠阴茎组织中nNOS的影响

目的：探讨针刺对心理应激性勃起功能障碍（ED）大鼠勃起功能影响的作用机制。方法：应用悬吊水池法建立心理应激性勃起功能障碍大鼠模型30只,将制模成功的大鼠随机分为模型组（n=10）、针刺组（n=10）,另设正常对照组（n=10）。针刺组取“关元”、“三阴交”、“肾俞”穴施治,应用免疫组化SP法检测3组大鼠阴茎组织中神经型一氧化氮合酶（nNOS）的表达。结果：与模型组相比,针刺能明显提高针刺组大鼠的勃起次数（P〈0．05）,并提高大鼠阴茎组织中nNOS的表达（P〈O．05）。结论：针刺能明显增强心理应激性勃起功能障碍大鼠的勃起功能,其作用机制与大鼠阴茎组织中nNOS水平升高有关。     

nNOS抑制剂亚胺基烯丁基-L-鸟氨酸对心肌缺血再灌注损伤的影响及机制

目的 探讨nNOS选择性抑制剂亚胺基烯丁基-L-鸟氨酸（L-VNIO）对心肌缺血再灌注（I/R）损伤的影响及机制。方法 构建SD大鼠离体心脏I/R模型和H9c2细胞缺氧/复氧（H/R）模型;nNOS抑制剂L-VNIO（10 μmol·L^-1）持续给药整个再灌注或复氧过程。TTC染色测定心肌梗死面积;流式细胞术检测H9c2细胞凋亡率;Fluo-3/AM Ca²⁺荧光探针通过流式细胞仪检测H9c2细胞内Ca²⁺浓度;试剂盒法测定离体心脏灌流液乳酸脱氢酶（LDH）、丙二醛（MDA）水平以及H9c2细胞MDA水平和超氧化物歧化酶（SOD）活性;离体心脏提取肌浆网,试剂盒法检测肌浆网Ca²⁺-ATP酶（SERCA）活性,Western blotting检测肌浆网SERCA蛋白表达;Western blotting检测离体心脏中受磷蛋白（PLB）和兰尼碱受体2（RyR2）蛋白表达水平和磷酸化水平。结果 与I/R或H/R模型组相比,L-VNIO显著降低细胞凋亡率,减少心肌梗死面积,降低LDH、MDA水平,提高SOD活性,差异均有统计学意义（P<0.05）;此外,与I/R或H/R模型组相比,L-VNIO组明显降低细胞内Ca²⁺超载,增高PLB磷酸化水平,降低RyR2磷酸化水平,增强SERCA活性（P<0.05）。结论 nNOS抑制剂L-VNIO可以减轻I/R损伤,机制与调节Ca²⁺转运相关蛋白而降低I/R引起的Ca²⁺超载相关。     

Embryo-fetal development toxicity of honokiol microemulsion intravenously administered to pregnant rats

The aim of this study was to evaluate the embryo-fetal development toxicity of honokiol microemulsion. The drug was intravenously injected to pregnant SD rats at dose levels of 0, 200, 600 and 2000 μg/kg/day from day 6–15 of gestation. All the pregnant animals were observed for body weights and any abnormal changes and subjected to caesarean-section on gestation day (GD) 20; all fetuses obtained from caesarean-section were assessed by external inspection, visceral and skeletal examinations. No treatment-related external alterations as well as visceral and skeletal malformations were observed in honokiol microemulsion groups. There was no significant difference in the body weight gain of the pregnant rats, average number of corpora lutea, and the gravid uterus weight in the honokiol microemulsion groups compared with the vehicle control group. However, at a dose level of 2000 μg/kg/day, there was embryo-fetal developmental toxicity observed, including a decrease in the body length and tail length of fetuses. In conclusion, the no-observed–adverse-effect level (NOAEL) of honokiol microemulsion is 600 μg/kg/day, 75 times above the therapeutic dosage and it has embryo-fetal toxicity at a dose level of 2000 μg/kg/day, which is approximately 250 times above the therapeutic dosage.     

石榴皮鞣质对大鼠乙醇性胃黏膜损伤的保护作用研究

目的:研究石榴皮鞣质对大鼠乙醇性胃黏膜损伤的保护作用。方法:乙醇灌胃(1.5mL/只)以复制大鼠乙醇性胃黏膜损伤模型。实验分为正常(等容生理盐水)、模型(等容生理盐水)、枸橼酸铋钾(100mg·kg-1)和石榴皮鞣质高、中、低剂量(500、150、50mg·kg-1)组,观察对胃黏膜的影响;测定一氧化氮(NO)、丙二醛(MDA)含量;通过免疫组化法测定神经元型一氧化氮合酶(nNOS)、内皮型一氧化氮合酶(eNOS)的表达。结果:150、500mg·kg-1石榴皮提取物可显著修复胃黏膜损伤,且可显著抑制模型大鼠MDA含量增高和NO含量降低(P<0.01),也可抑制nNOS、eNOS的表达下降,但无显著性差异。结论:石榴皮提取物对大鼠乙醇性胃黏膜损伤具有良好的保护作用,这种作用可能与抗脂质过氧化反应,抑制nNOS、eNOS表达下降有关。     

Cyclo(dehydrohistidyl-l-tryptophyl) inhibits nitric oxide production by preventing the dimerization of inducible nitric oxide synthase

Dimerization of inducible NOS has been known to be a potential therapeutic target for iNOS-mediated pathologies. Cyclic dipeptides are among the simplest peptides commonly found as by-products of food processing or metabolites of microorganisms. In this study, we found that cyclo(dehydrohistidyl-l-tryptophyl) (CDHT), a cyclic dipeptide from an unidentified fungal strain Fb956, prevents iNOS dimerization in activated microglial BV-2 cells. CDHT inhibited NO production with an IC50 of 6.5 microM in LPS-treated BV-2 cells. Western blot analysis and iNOS activity measurement of fractions from size-exclusion chromatography of cell lysates indicated that CDHT inhibits dimerization of iNOS, while it has no effect on iNOS expression or enzyme activity. The CDHT inhibition of iNOS dimerization was confirmed by partially denaturing SDS-PAGE analysis. In contrast, CDHT did not affect cGMP production in endothelial HUVEC cells, which indicates no inhibition of endothelial NOS activity. These results reveal that CDHT, one of the simplest and cyclic dipeptides, selectively inhibits NO production by inhibiting iNOS dimerization, and could be a useful therapeutic agent for inflammation-mediated diseases.     

Spinal nerve ligation reduces nitric oxide synthase activity and expression: effect of resveratrol

The effect of resveratrol on activity and expression of nitric oxide synthase (NOS) in the spinal cord of neuropathic rats was assessed. Spinal nerve ligation produced tactile allodynia along with a reduction of catalytic activity of the constitutive Ca²⁺-dependent NOS (eNOS and nNOS isoforms) in the ipsilateral dorsal horn, but not contralateral dorsal or ipsilateral or contralateral ventral, spinal cord at 1, 5, 10 and 15 days after surgery compared to naïve and sham-operated animals. Nerve ligation also induced a reduction of nNOS expression in the ipsilateral dorsal horn spinal cord at 10 and 15 days after surgery. Intrathecal resveratrol reduced allodynia and reversed the reduction of constitutive Ca²⁺-dependent NOS activity in the ipsilateral dorsal spinal cord. Moreover, resveratrol significantly reversed the reduction of nNOS expression in the ipsilateral dorsal horn spinal cord. Results show that spinal nerve ligation leads to development of tactile allodynia along with a reduction in constitutive Ca²⁺-dependent NOS activity and nNOS isoform expression in the ipsilateral dorsal horn. Data suggest that resveratrol may reduce tactile allodynia in neuropathic rats by restoring altered NOS activity and expression.